Abstract
The recently synthesized isocyanonaphtalene derivatives ACAIN and CACAIN are fluorochromes excitable at wavelengths of around 366 nm and bind cysteine-rich proteins with hydrophobic motifs. We show that these compounds preferentially label tonoplasts in living Arabidopsis and tobacco (Nicotiana tabacum SR1) cells. ACAIN-labeled membranes co-localized with the GFP signal in plants expressing GFP-δ-TIP (TIP2;1) (a tonoplast aquaporin) fusion protein. ACAIN preserved the dynamics of vacuolar structures. tip2;1 and triple tip1;1-tip1;2-tip2;1 knockout mutants showed weaker ACAIN signal in tonoplasts. The fluorochrome is also suitable for the labeling and detection of specific (cysteine-rich, hydrophobic) proteins from crude cell protein extracts following SDS-PAGE and TIP mutants show altered labeling patterns; however, it appears that ACAIN labels a large variety of tonoplast proteins. ACAIN/CACAIN could be used for the detection of altered vacuolar organization induced by the heptapeptide natural toxin microcystin-LR (MCY-LR), a potent inhibitor of both type 1 and 2A protein phosphatases and a ROS inducer. As revealed both in plants with GFP-TIP2;1 fusions and in wild-type (Columbia) plants labeled with ACAIN/CACAIN, MCY-LR induces the formation of small vesicles, concomitantly with the absence of the large vegetative vacuoles characteristic for differentiated cells. TEM studies of MCY-LR-treated Arabidopsis cells proved the presence of multimembrane vesicles, with characteristics of lytic vacuoles or autophagosomes. Moreover, MCY-LR is a stronger inducer of small vesicle formation than okadaic acid (which inhibits preferentially PP2A) and tautomycin (which inhibits preferentially PP1). ACAIN and CACAIN emerge as useful novel tools to study plant vacuole biogenesis and programmed cell death.
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References
Allen MM (1968) Simple conditions for the growth of unicellular blue-green algae on plates. J Phycol 4(1):1–4. https://doi.org/10.1111/j.1529-8817.1968.tb04667.x
Batistič O (2012) Genomics and localization of the Arabidopsis DHHC-cysteine-rich-domain S-acyltransferase protein family. Plant Physiol 160(3):1597–1612. https://doi.org/10.1104/pp.112.203968
Beebo A, Thomas D, Der C, Sanchez L, Leborgne-Castel N, Marty F, Schoefs B, Bouhidel K (2009) Life with and without AthTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles. Plant Mol Biol 70(1-2):193–209. https://doi.org/10.1007/s11103-009-9465-2
Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15(3):441–447. https://doi.org/10.1046/j.1365-313X.1998.00208.x
Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214(2):159–173. https://doi.org/10.1111/j.0022-2720.2004.01348.x
Bouaïcha N, Maatouk I (2004) Microcystin-LR and nodularin induce intracellular glutathione alteration, reactive oxygen species production and lipid peroxidation in primary cultured rat hepatocytes. Toxicol Lett 148(1-2):53–63. https://doi.org/10.1016/j.toxlet.2003.12.005
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Campos A, Vasconcelos VM (2010) Molecular mechanisms of microcystin toxicity in animal cells. Intl J Mol Sci 11(1):268–287. https://doi.org/10.3390/ijms11010268
Chen L, Xie P (2016) Mechanisms of microcystin-induced cytotoxicity and apoptosis. Mini-Rev Med Chem 16(13):1018–1031. https://doi.org/10.2174/1389557516666160219130407
Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci U S A 97(7):3718–3723. https://doi.org/10.1073/pnas.97.7.3718
Emans N, Zimmermann S, Fischer R (2002) Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin. Plant Cell 14(1):71–86. https://doi.org/10.1105/tpc.010339
Feraru E, Paciorek T, Feraru MI, Zwiewka M, De Groodt R, De Rycke R, Kleine-Vehn J, Friml J (2010) The AP-3 b adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis. Plant Cell 22(8):2812–2824. https://doi.org/10.1105/tpc.110.075424
Foresti O, daSilva LLP, Denecke J (2006) Overexpression of the Arabidopsis syntaxin PEP12/SYP21 inhibits transport from the prevacuolar compartment to the lytic vacuole in vivo. Plant Cell 18(9):2275–2293. https://doi.org/10.1105/tpc.105.040279
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158. https://doi.org/10.1016/0014-4827(68)90403-5
Gao C, Zhuanga X, Cuia Y, Fua X, Hea Y, Zhaoa Q, Zenga Y, Shena J, Luoa M, Jiang L (2015) Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 112(6):1886–1891. https://doi.org/10.1073/pnas.1421271112
Kós P, Gorzó G, Surányi G, Borbely G (1995) Simple and efficient method for isolation and measurement of cyanobacterial hepatotoxins by plant tests (Sinapis alba L.) Anal Biochem 225(1):49–53. https://doi.org/10.1006/abio.1995.1106
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0
Liu Y, Bassham DC (2012) Autophagy: pathways for self-eating in plant cells. Annu Rev Plant Biol 63(1):215–237. https://doi.org/10.1146/annurev-arplant-042811-105441
Liu Y, Schiff M, Czimmek K, Tallóczy Z, Levine B, Dinesh-Kumar SP (2005) Autophagy regulates programmed cell death during the plant innate immune response. Cell 121(4):567–577. https://doi.org/10.1016/j.cell.2005.03.007
Liu Y, Burgos JS, Deng Y, Srivastava R, Howell SH, Basshama DC (2012) Degradation of the endoplasmic reticulum by autophagy during endoplasmic reticulum stress in Arabidopsis. Plant Cell 42(11):4635–4651. https://doi.org/10.1105/tpc.112.101535
Maeshima M (2001) Tonoplast transporters: organization and function. Annu Rev Plant Physiol Plant Mol Biol 52:469–497
Martinoia E, Massonneau A, Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plants. Plant Cell Physiol 41(11):1175–1186. https://doi.org/10.1093/pcp/pcd059
Máthé C, M-Hamvas M, Vasas G (2013) Microcystin-LR and cylindrospermopsin induced alterations in chromatin organization of plant cells. Mar Drugs 168(10):3689–3717. https://doi.org/10.3390/md11103689
Máthé C, Beyer D, M-Hamvas M, Vasas G (2016) The effects of microcystins (cyanobacterial heptapeptides) on the eukaryotic cytoskeletal system. Mini-Rev Med Chem 16(13):1063–1077. https://doi.org/10.2174/1389557516666160219130732
Mathur J, Mathur N, Kirik V, Kernebeck B, Srinivas BP, Hülskamp M (2003) Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation. Development 130(>14):3137–3146. https://doi.org/10.1242/dev.00549
Meckel T, Hurst AC, Thiel G, Homann U (2004) Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labeled plasma membrane and GFP-tagged K+ channel KAT1. Plant J 39(2):182–193. https://doi.org/10.1111/j.1365-313X.2004.02119.x
Moriyasu Y, Hattori M, Jauh GY, Rogers JC (2003) Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process. Plant Cell Physiol 44(8):795–802. https://doi.org/10.1093/pcp/pcg100
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nagy M, Rácz D, Nagy ZL, Nagy T, Fehér PP, Purgel M, Zsuga M, Kéki S (2016) An acrylated isocyanonaphthalene based solvatochromic click reagent: optical and biolabeling properties and quantum chemical modeling. Dyes Pigments 133:445–457. https://doi.org/10.1016/j.dyepig.2016.06.036
Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51(6):1126–1136. https://doi.org/10.1111/j.1365-313X.2007.03212.x
Oparka KJ (1994) Plasmolysis: new insights into an old process. New Phytol 126(4):571–591. https://doi.org/10.1111/j.1469-8137.1994.tb02952.x
Reinhardt H, Hachez C, Bienert MD, Beebo A, Swarup K, Voß U, Bouhidel K, Frigerio L, Schjoerring JK, Bennett MJ, Chaumont F (2016) Tonoplast aquaporins facilitate lateral root emergence. Plant Physiol 170:1640–1654. https://doi.org/10.1104/pp.15.01635
Schüssler MD, Alexandersson E, Bienert GP, Kichey T, Laursen KH, Johanson U, Kjellbom P, Schoellring JF, Jahn TP (2008) The effects of the loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana. Plant J 56(5):756–767. https://doi.org/10.1111/j.1365-313X.2008.03632.x
Swanson SJ, Bethke PC, Jones RL (1998) Barley aleurone cells contain two types of vacuoles: characterization of lytic organelles by use of fluorescent probes. Plant Cell 10(5):685–698. https://doi.org/10.1105/tpc.10.5.685
Swingle M, Ni L, Honkanen RE (2007) Small molecule inhibitors of Ser/Thr protein phosphatases: specificity, use and common forms of abuse. Methods Mol Biol 365:23–38. https://doi.org/10.1385/1-59745-267-X:23
Vasas G, Gáspár A, Páger C, Surányi G, Máthé C, M-Hamvas M, Borbély G (2004) Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-LR) by capillary electrophoresis. Electrophoresis 25(1):108–115. https://doi.org/10.1002/elps.200305641
Zhao J, Dixon RA (2009) MATE transporters facilitate vacuolar uptake of epicatechin 3′-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatula and Arabidopsis. Plant Cell 21(8):2323–2340. https://doi.org/10.1105/tpc.109.067819
Zwiewka M, Feraru E, Möller B, Hwang I, Feraru MI, Kleine-Vehn J, Weijers D, Friml J (2011) The AP-3 adaptor complex is required for vacuolar function in Arabidopsis. Cell Res 21(12):1711–1722. https://doi.org/10.1038/cr.2011.99
Acknowledgements
This work was financially supported by the grants K-116465 and K-120638 given by NKFIH (National Research, Development and Innovation Office, Hungary) and GINOP-2.3.2-15-2016-00041 and GINOP-2.3.3.-15-2016-00030 project. The project is co-financed by the European Union and the European Regional Development Fund. CM was supported by the Balassi Institute/Campus Hungary Mobility Support No. B2/2H/7717 for a mobility to the University of Guelph, ON, Canada in 2014. MN was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. JM acknowledges a Discovery Grant from NSERC, Canada. We would like to thank Prof. Ferenc Erdődi for providing us with okadaic acid and tautomycin and Sean Cutler for the GFP-TIP2;1 transgenic Arabidopsis line.
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Supplementary Movie 1
Normal vacuolar dynamics in a control GFP-TIP2;1 plant labeled with ACAIN: composite ACAIN labeling and GFP signal are seen (AVI 3890 kb)
Supplementary Movie 2
Vacuolar dynamics in a GFP-TIP2;1 plant treated for 4 h with 1 μM MCY-LR, GFP signal is seen. The absence of large vacuoles and impaired dynamics of tonoplast-coated vesicles are seen (AVI 3381 kb)
Supplementary Fig. 1
a–c ACAIN does not label ER and the Golgi apparatus as shown by the different distribution patterns of fluorescence labeling for these two membrane compartments in Arabidopsis hypocotyls. a EYFP-ER plants (Nelson et al. 2007) showing the ER network and bright yellow ER bodies; b GFP-ERD2 plants (Boevink et al. 1998; Mathur et al. 2003) showing labeling of Golgi (green) and chlorophyll autofluorescence in plastids (blue); c ACAIN labeling of tonoplasts. LSM excitation and emission settings were the characteristic GFP and YFP settings of the Leica TCS-SP5 confocal microscope with the use of the HCX APO L U-V-I 40.0 × 0.80 water immersion objective for (a, b) and the Zeiss LSM 880 confocal microscope setting for ACAIN labeling (c); they were as specified in the “Materials and methods” section. d, e ACAIN labeling of plasmolyzed hypocotyl cells from YFP-PIP2a plants (Cutler et al. 2000). Plasmolysis was done with 5% (w/v) NaCl for 5 min. These images show that ACAIN labels preferentially the tonoplast and not the plasma membrane. Merged images show both the ACAIN and YFP label as well as the brightfield background; the latter is for showing cell contours. e Detail of the merged image of (d). H, Hechtian strands showing only the YFP label; v, shrunk vacuoles showing only the ACAIN label. GFP and YFP settings were those of the Zeiss LSM 880. Scale bars: 30 μm (JPEG 13046 kb)
Supplementary Fig. 2
Preparation of Col-0 protein extracts in the standard Laemmli buffer without boiling gives sufficient protection against proteolytic degradation as shown by both ACAIN labeling and Coomassie Blue staining. A1—extract prepared in the presence of 0.5% (v/v) protease inhibitor cocktail without boiling; A2—extract prepared without protease inhibition and boiling; A3—extract prepared without protease inhibition, but boiled; M—position of the bands of molecular weight marker (JPEG 1686 kb)
Supplementary Fig. 3
a TEM image of a hypocotyl cell treated for 4 days with 1 μM MCY-LR showing a multimembrane vesicle (arrow) after its incorporation into the central vacuole (CV). b TEM image of autophagosome-like structures (a) inside a large vacuole of a hypocotyl cortex cell treated with 1 μM MCY-LR for 3 days. Scale bars: 1 μm (JPEG 2731 kb)
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Nagy, M., Kéki, S., Rácz, D. et al. Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors. Protoplasma 255, 829–839 (2018). https://doi.org/10.1007/s00709-017-1190-0
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DOI: https://doi.org/10.1007/s00709-017-1190-0